Chrono-Environnement Research Team UMR/CNRS-6249-Franche-Comté University, Besançon, France; Department of Parasitology and Mycology, University Hospital, Besançon, France.
Sci Total Environ. 2014 Jan 1;466-467:716-24. doi: 10.1016/j.scitotenv.2013.07.054. Epub 2013 Aug 25.
The aim of the present study was to assess performance, feasibility and relevance of a Standard Operational Procedure (SOP) for large-scale use in the microbial analysis of children's indoor environments. We analyzed dust settled on Electrostatic Dust Fall Collectors (EDCs) by using qPCR which targeted 6 molds, 3 bacteria and 1 mite, chosen for their involvement in allergic or inflammatory processes. Six types of commercialized electrostatic wipes were tested for their releasing capacity of fungal DNA from fungal spores captured by the wipes. Specificity, repeatability and detection limits of the qPCR procedure were tested using calibrated microbial suspensions. The feasibility and relevance of this sampling and analysis method were assessed in a 75-home pilot study. Our result showed that one specific make of wipe was more effective than the others in releasing fungal DNA. qPCR procedure showed good repeatability. The quantification limit was about 5 fg DNA/μL for all species except Penicillium chrysogenum (0.5 fg DNA/μL) and Dermatophagoïdes pteronyssinus (10 fg DNA/μL). No cross-reactivity was observed. DNA concentrations in the 53/75 homes participating in the pilot study were between 0 and 24 625, 0 and 69 738 equivalent cells per cm(2) for the fungi and bacteria, and between 0 and 1 equivalent mites per cm(2) for D. pteronyssinus. Using the SOP described, we were able to classify the 53 dwellings from the least to the most contaminated according to the quantity of DNA measured for each species. Our SOP measured fungi, bacteria and mites using a cost-efficient, discreet and well-accepted sampling method with just one qPCR tool. The whole procedure can be used for microbial analysis in large cohort studies such as the ELFE study ("Etude Longitudinale Française depuis l'Enfance") and could help improve our understanding of the interactions between the environment, allergic diseases and child development.
本研究旨在评估大规模使用静电尘埃收集器（EDC）采集儿童室内环境微生物样本的标准操作程序（SOP）的性能、可行性和相关性。我们使用 qPCR 分析了落在 EDC 上的灰尘，qPCR 靶向了 6 种真菌、3 种细菌和 1 种尘螨，这些真菌、细菌和尘螨都与过敏或炎症过程有关。我们测试了 6 种商业化的静电擦拭布释放拭子捕获的真菌孢子中真菌 DNA 的能力。使用校准的微生物悬浮液测试了 qPCR 程序的特异性、重复性和检测限。在一项 75 户的试点研究中评估了这种采样和分析方法的可行性和相关性。结果显示，一种特定的擦拭布比其他擦拭布更有效地释放真菌 DNA。qPCR 程序显示出良好的重复性。除青霉（Penicillium chrysogenum）（0.5 fg DNA/μL）和嗜皮菌（Dermatophagoïdes pteronyssinus）（10 fg DNA/μL）外，所有物种的定量限约为 5 fg DNA/μL。未观察到交叉反应。参与试点研究的 53 户家庭中，有 53 户家庭的真菌和细菌 DNA 浓度在 0 到 24625 之间，真菌和细菌的等效细胞数在 0 到 69738 之间，D. pteronyssinus 的尘螨数在 0 到 1 之间。使用所描述的 SOP，我们能够根据每种物种测量的 DNA 量将 53 个住宅从污染最少到污染最多进行分类。我们的 SOP 使用一种成本效益高、隐蔽性好、易于接受的采样方法，仅使用一种 qPCR 工具测量真菌、细菌和尘螨。整个过程可用于大规模队列研究（如 ELFE 研究）中的微生物分析，有助于提高我们对环境、过敏疾病和儿童发育之间相互作用的理解。
